How to Optimize Your Multiplex Testing for Diagnostics Applications
Original article written and published by Today's Clinical Lab.
Gerald (Jerry) Hunter, PhD, is a former field application scientist of Fortis Life Sciences with extensive experience in developing nucleic acid extraction, PCR, and many molecular biology techniques.
Custom reagents for multiplex PCR assays may be the missing ingredients for your infectious disease panel.
How does multiplex testing fit into the infectious disease diagnostics landscape?
Multiplex testing allows you to detect multiple analytes or loci from different pathogens within a single well. This is advantageous for labs because you can get more data out of doing less, saving time and reagents by running one experiment rather than testing one-by-one for each pathogen.
While there is still interest in using multiplex PCR for COVID-19, influenza A and B, and RSV (respiratory syncytial virus) testing, multiplex testing is also used for UTIs (urinary tract infections) and STIs (sexually transmitted infections).
What are the key considerations for designing a multiplex PCR experiment?
Other than the considerations with traditional PCR, including the correct concentrations of buffers, dNTPs, and magnesium, for multiplex workflows, we emphasize using a hot start DNA polymerase. Since this requires a temperature of 95°C, users can set up their experiment at the benchtop instead of on ice, avoiding off-target amplification until the temperature is ramped up within the thermocycler. Given that multiplex assays amplify multiple targets, you also need to scale up reagent concentrations to accommodate all of that amplification.
What are the key considerations for verifying and validating a multiplex PCR assay?
There are many considerations in analytical detection experiments like multiplex PCR, including the following:
- Accuracy: Are you amplifying the nucleic acid target you want to amplify?
- Precision: Are you amplifying the right region of your DNA or RNA to successfully identify whether the sample is positive or negative for a given pathogen?
- Dynamic range: What is the assay’s range in terms of how high and low the concentration can be such that clinical samples always fall within that range?
- Sensitivity and specificity: How little of a sample can you use and still amplify the correct target regions? Sensitivity is important for clinical samples because they are difficult to acquire in large volumes, and you have limited opportunity to collect more of the sample.
To optimize for these considerations, clinical labs run a couple of hundred samples in multiple PCR plates with different parameters to check for reproducible results between replicates.
How can working with a contract manufacturing organization (CMO) help clinical labs develop and implement effective multiplex PCR assays?
Not all labs are going to be able to find what they need for their multiplex assay in a catalog. Many labs need customized solutions in terms of the volume, the concentration of specific components, and the format of reagents. For example, in point-of-care settings, we see a demand for lyophilization, where instead of a "wet chemistry" format, all the assay components are dried into a bead. Adding your sample reconstitutes that bead, and then you can amplify your nucleic acid targets.
CMOs like Fortis can provide these custom solutions. We have the internal expertise, workflow, ISO certification, and scaling capabilities to help labs apply our custom reagents, resulting in multiplex assays optimized for accuracy, precision, range, sensitivity, and specificity.
